Chimeric protein for prevention and treatment of HIV infection

ABSTRACT

This invention relates to bispecific fusion proteins effective in viral neutralization. More specifically, such proteins have two different binding domains, an inducing-binding domain and an induced-binding domain, functionally linked by a peptide linker. Such proteins, nucleic acid molecules encoding them, and their production and use in preventing or treating viral infections are provided. One prototypical bispecific fusion protein is sCD4-SCFv(17b), in which a soluble CD4 fragment (containing domains D1 and D2) is fused to a single chain Fv portion of antibody 17b via a linker.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the National Stage of International Application No.PCT/US00/06946, filed Mar. 16, 2000, and claims the benefit of U.S.Provisional Application No. 60/124,681, filed Mar. 16, 1999. Theprovisional application is incorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates to proteins useful in the prevention andtreatment of human immunodeficiency virus (HIV) infection. Morespecifically, it relates to fusion proteins that bind to two sites on asingle target protein, especially when one binding domain of the fusionprotein binds to an induced site (on the target protein) that is exposedby the binding of the other binding domain of the fusion protein.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is a fatal disease of growingprevalence in the modern world. The agent responsible for this disease,human immunodeficiency virus (HIV), was first identified in 1983. HIV isa T-lymphotropic retrovirus that invades and replicates in cells of theimmune system, primarily helper T-lymphocytes. The consequentdysfunction in T-lymphocyte-mediated immunity results in animmuno-compromised condition. Patients usually die of associatedopportunistic viral, bacterial or fungal infections. A characteristicslaboratory finding in AIDS is the decrease in helper T lymphocytes(CD4), and particularly a steady decrease in the ratio of CD4 tosuppressor T lymphocytes CD8 as the disease progresses. Virus binding isprimarily mediated by interaction of gp120, the external subunit of theHIV envelope glycoprotein (Env) with CD4 protein and various coreceptormolecules (one of several alternative chemokine receptors). Theseinteractions then activate the gp41 transmembrane subunit of theenvelope glycoprotein, to cause fusion between the virus and cellmembranes. See Retroviruses, Coffin et al. (eds.) (1997) CSHP, New York,Ch. 11.

The humoral immune system is triggered by HIV infection, though itgenerally does not provide sufficient protection to ward off theinfection. Env is the major target of anti-HIV neutralizing antibodies(Wyatt et al. Nature 393:705–711, 1998). However, Env has evolved sothat its relatively invariant neutralizing determinants are protectedfrom the humoral immune system. Antibodies to these regions thereforeare generated at a low frequency and their neutralizing activities invivo are generally weak. Certain variable regions (e.g., the V3 loop)are targets for potent neutralizing antibodies, but these are typicallyrestricted to a limited number of HIV-strains (in other words, they arenot broadly cross-reactive). For a list of several gp120 antigenicepitopes and consensus definitions of the conserved and variable regionsof gp120, see published PCT application PCT/US98/02766 (publicationnumber WO 98/36087) and Coffin et al. (eds.) (1997) CSHP, New York, Ch.12.

A neutralizing monoclonal antibody (MAb) with potent and broadlycross-reactive activity would have great potential value in protocolsaimed at preventing HIV infection before or immediately after exposure,for example in neonatal transmission, post-exposure prophylaxis, and asa topical inhibitor. Such a MAb may also be useful in treating chronicinfection (D'Souza et al. J. Infect. Dis. 175:1056–1062, 1997). Howeveronly a handful of MAbs with the desired broadly cross-reactiveneutralizing activities have been described. Because of limited potencyand cross-reactivity of these molecules, even the three most promisingcandidates have questionable clinical value (D'Souza et al., 1997).

Extensive efforts are underway to provide immunological orpharmacological approaches to controlling HIV infection (Coffin et al.,1997, Ch. 12). The specific interaction between gp120 and CD4 has beenexploited in efforts to provide a possible treatment for HIV infection.See, e.g., U.S. Pat. No. 5,817,767; Capon et al., Nature 337:525–531,1989. A soluble fragment of CD4 (sCD4), comprising the first and seconddomains of this protein (D1 D2) has been generated, and this moleculeinteracts specifically with gp120, essentially serving as a moleculardecoy. sCD4 has been shown to block the spread of HIV between culturedcells (Moore et al., Science 250:1139–1142, 1990). However, clinicaltrials with sCD4 were inconclusive as to the effects on human viral load(Schooley et al., Ann. Internal Med. 112:247–253, 1990; Kahn et al.,Ann. Internal Med. 112:254–261, 1990). Subsequent studies indicatedthat, unlike laboratory-adapted HIV strains, isolates obtained directlyfrom infected patients (primary isolates) are resistant toneutralization by sCD4 (Darr et al., Proc. Natl. Acad. Sci.87:6574–6578, 1990).

In another approach, researchers have generated an antibody-likemolecule by fusing the binding portion of CD4 to the constant region(Fc) of a human IgG heavy chain (see, e.g., Capon et al., Nature337:525–531, 1989; and Byrn et al., Nature 344:667–670, 1990). Thismolecule, termed CD4 immunoadhesin, exploits the native functions ofimmunoglobulin Fc, such as its ability to fix complement, its ability tomediate antibody-dependent cytotoxicity, and its transfer across theplacental barrier. There are significant drawbacks to using Fc receptorsin association with CD4, because such a construct may be responsible fortargeting HIV to Fc-receptor bearing cells (e.g. macrophages), and mightlead to increased transmission of HIV-1 across the placental barrier.

A complementary recombinant molecule has also been made, wherein thebinding portion of CD4 is fused to the Fv region of an antibody directedto human CD3; this “Janusin” molecule may be able to re-target cytotoxicT-lymphocytes onto HIV-infected cells (Traunecker et al., Embo J.10:3655–3659, 1991; Traunecker et al., Int. J. Cancer: Supp. 7:51–52,1992). Janusin has been reported to inhibit HIV-mediated cell fusionwhen administered in vito with neutralizing antibody to either gp41 orthe V3 loop of gp120 (Allaway et al., AIDS Res. Hum. Retroviruses9:581–587, 1993; U.S. Pat. No. 5,817,767). This system is inherentlycomplicated and inefficient because multiple molecules must beco-administered to the subject.

This invention is directed to proteins that address key failures of theprior art.

SUMMARY OF THE INVENTION

The present invention takes advantage of the finding that theneutralizing activities of MAbs against certain highly conserveddeterminants of the coreceptor-binding region of gp120 are revealed onlywhen CD4 first binds to gp120 (as in an sCD4-activated fusion assay).Although some MAbs to CD4-induced epitopes (e.g., the human MAbs 17b and48d, Thali et al., J. Virol. 67:3978–3988, 1993) are broadlycross-reactive with Envs from diverse HIV genetic subtypes (Clades),these neutralizing epitopes are only briefly exposed in vivo, andtherefore have provided poor targets for clinically protective antibodybinding.

The inventors have overcome these difficulties by creating a fusionprotein containing a fragment of CD4 attached via a linker to a humansingle chain Fv directed against an induced (for example, a CD4-induced)neutralizing epitope on gp120, for instance a coreceptor-bindingdeterminant of gp120. CD4-binding exposes highly conserved gp120determinants involved in binding to coreceptor; therefore the providedfusion protein will have the properties of a highly potent, broadlycross-reactive neutralizing antibody with high in vivo activity and noFc-mediated undesirable targeting properties. When the fusion protein issubstantially derived from human proteins, it has minimal immunogenicityand toxicity in humans. Such an agent has great value in the preventionof infection during or immediately after HIV exposure (mother/infanttransmission, post-exposure prophylaxis, topical inhibitor), and also inthe treatment of chronic infection.

Accordingly, a first embodiment of the current invention is aneutralizing bispecific fusion protein capable of binding to two siteson a target protein. This protein has two different binding domains, aninducing-binding domain and an induced-binding domain, functionallylinked by a peptide linker. Nucleic acid molecules encoding such fusionproteins are further aspects of this invention. Also encompassed in theinvention are protein analogs, derivatives, or mimetics of suchneutralizing bispecific fusion proteins. The arrangement of theinducing- and induced-binding domains need not be organized in bindingsequence; the amino-proximal or carboxy-proximal binding domain of thefusion protein may be either the induced-binding or the inducing-bindingdomain.

In certain embodiments, the linker of this invention is of such lengthand secondary structure that the linker allows the second binding domainto be in binding proximity to the induced epitope of the target proteinwhen the first binding domain is bound to the inducing site of thetarget protein. The linker may for instance be substantially flexible.Linkers of about 25–100 angstroms (Å), or about 15–100 amino acidresidues in length, are examples of linkers of a sufficient length tomaintain the second binding domain in binding proximity to the inducedepitope. Specific examples of linkers will include one or moreoccurrences of the amino acid sequence represented by SEQ ID NO: 1. Forinstance, the invention encompasses bispecific fusion proteins whereinthe two binding domains are functionally linked by the amino acidsequence represented by SEQ ID NO: 2.

Targets for bispecific fusion proteins according to this inventioninclude viral envelope proteins. For instance, viral envelope proteinsfrom the human immunodeficiency virus (HIV) are targets for thedisclosed invention. In a specific embodiment of the invention, theviral envelope protein target is gp120.

In further aspects of the invention, the first binding domain is capableof binding to an inducing site on the target protein, thereby exposingan induced epitope. For instance, the first binding domain can be aligand such as CD4 or fragments thereof. Alternatively, such a firstbinding domain may be a binding portion of a variable region of anantibody heavy or light chain. The first binding domain may include, forexample, an antibody-binding domain, a single-chain Fv (SCFv), orbinding fragments thereof.

The second binding domain, which is capable of forming a neutralizingcomplex with an induced epitope of the target protein, may be forexample an antibody or fragments thereof, such as the variable region,Fv, Fab or antigen-binding domain of an antibody. Another example of thesecond binding domain of the fusion protein is an engineeredsingle-chain Fv (SCFv).

In some particular examples where HIV gp120 is the target, and theinducing site is the gp120 CD4 binding site, the induced epitope may bea coreceptor-binding determinant of gp120. Accordingly, aspects of thisinvention include proteins in which the first binding domain binds togp120 in such a way as to cause a CD4-induced conformational change inthe complexed gp120 that exposes the second binding domain. The firstbinding domain may be derived from a CD4 molecule, and include CD4 andsoluble fragments thereof (sCD4, e.g. D1, D1D2 and other suchfragments), and proteins that mimic the biological activity of a CD4molecule in binding to the inducing site of gp120. In another embodimentof the invention, the first domain of the gp120-targeted bispecificfusion protein is derived from a CD4 anti-idiotypic antibody, orantibodies that mimic CD4 in exposing epitopes.

The second domain of the gp120-targeted bispecific fusion protein, whichbinds to an epitope induced by binding of the first fusion domain, maybe chosen from domains and fragments of proteins that bind to such CD4induced epitopes. Antibodies directed to the induced epitopes, as wellas the HIV coreceptor (e.g. a chemokine receptor), HIV coreceptormimics, and fragments of HIV coreceptor proteins, are examples ofsources for the second binding domain of a gp120-target bispecificfusion protein of this invention. Examples of chemokine receptors withHIV coreceptor activity include CXCR4, CCR5, CCR2B, and CCR3.Neutralizing antibodies, including 17b and 48d, are examples ofantibodies. Fusion proteins wherein the second domain is an engineeredsingle chain Fv (SCFv) derived from such a neutralizing antibody arealso encompassed.

A particular embodiment of this invention is a functional recombinantbispecific fusion protein capable of binding to two sites on gp120,wherein the inducing-binding domain is sCD4; the induced-binding domainis SCFv(17b); and these two domains are linked by a linker of a lengthsufficient to maintain the SCFv(17b) in binding proximity an SCFv(17b)epitope when sCD4 is bound to gp120. A prototypical bispecific fusionprotein has the amino acid sequence shown in SEQ ID NO: 3. Nucleic acidmolecules encoding such a fusion protein are also encompassed; theprototypical nucleic acid molecule has the sequence shown in SEQ ID NO:4. Vectors and cells comprising this nucleic acid molecule are alsoencompassed in the current invention, as are transgenic plants andanimals that express the nucleic acid molecule.

This invention also provides methods for producing functionalrecombinant bispecific fusion proteins capable of binding two sites on atarget protein. Such a protein can be produced in a prokaryotic oreukaryotic cell (e.g., yeast, insect and mammalian cells), for instanceby transforming or transfecting such a cell with a recombinant nucleicacid molecule comprising a sequence which encodes a disclosed bispecificfusion protein. Such transformed cells can then be cultured underconditions that cause production of the fusion protein, which is thenrecovered through protein purification means. The protein can include amolecular tag, such as a six-histidine tag, to facilitate its recovery.In particular embodiments, the protein has a hexa-histidine (hexa-his)tag, and a thrombin cleavage site.

The invention further provides methods for inactivating a targetprotein, for instance a gp120 protein, by contacting the target proteinwith a fusion protein according to this invention. Where the targetprotein is gp120, this method involves contacting gp120 with agp120-targeted bispecific fusion protein, for instance sCD4-SCFv(17b).Proteins according to the current invention can also be used toneutralize a human immunodeficiency virus, by contacting the humanimmunodeficiency virus with a gp120-targeted fusion protein according tothis invention. Binding of a viral or recombinant gp120 protein tosoluble CD4 or lymphocyte CD4 can also be blocked and/or prevented bycontacting the gp120 protein with gp120-targeted fusion protein. In anyof these methods, a variant protein, analog or mimetic of the fusionprotein as provided herein may also be used.

Proteins of the current invention can be used to inhibit virusreplication or infectivity in a subject by administering to the subjectan amount of the fusion protein (for example the sCD4-SCFv(17b) fusionprotein), or a variant protein, analog or mimetic thereof, sufficient toinhibit HIV virus replication or infectivity. The fusion protein can beadministered in a pharmaceutical composition, and given therapeuticallyto a person who is known to be infected with HIV, or prophylactically tohelp prevent infection in someone who has been exposed to the virus, oris at high risk for exposure. Proteins of this invention can also beadministered in combination with another compound for the treatment orprevention of HIV infection, such as an HIV reverse transcriptase (RT),integrase, or protease inhibitor, another HIV-1 neutralizing antibody,or an Env-targeted toxin. The other drug may be an HIV antiviral agent,an HIV anti-infective agent, and/or an immunomodulator, or combinationsthereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph illustrating relative HIV-1 Env-mediated fusion,in the presence (+) or absence (−) of soluble CD4, between effectorcells expressing Env (Ba-L) and target cells expressing CCR5(co-receptor), but no CD4 (primary receptor).

FIG. 2 is a graph showing that antibody 17b does not inhibit HIV-1Env-mediated fusion in the conventional assay (open box: CXCR4 and CD4on target cell), but strongly inhibits cell fusion in the sCD4-activatedassay (filled circle: only CXCR4 on target cell, sCD4 provided).Additional experiments indicate that this phenomenon occurs with diverseEnvs using either CXCR4 or CCR5, and that 17b has broad cross-reactiveactivity with Envs from genetically diverse HIV-1 isolates.

FIG. 3 is a schematic diagram of the CD4-SCFv(17b) genetic construct.The genetic construct encodes sCD4 (D1D2, plus the native CD4 N-terminalsignal sequence), followed by the L1 linker (Gly₄Ser)₇, which attachesthe 17b SCFv (V_(H) attached to V_(L) via the L2 linker (Gly₄Ser)₃),followed by the thrombin cleavage site and hexa-his tag. There is a BamHI site in the middle of L1 to facilitate production of constructs ofdifferent lengths.

FIG. 4 is a drawing of mechanisms of binding of a sCD4-SCFv(17b) togp120, and the resulting neutralization of HIV Env function (fusion andinfectivity).

FIGS. 4A, 4B, and 4C depict the proposed interaction of HIV (mediated bygp120) with the cell surface receptor CD4 and co-receptor CCR5, and thebeginning of fusion (mediated by gp41). Interaction between gp120 andCD4 (FIG. 4A) causes a change in the conformation of gp120 (FIG. 4B),which enables interaction between gp120 and CCR5 (FIG. 4B). Thistriggers a conformational change in gp41 (FIG. 4C), and leads to fusion.Antibody (for instance, MAb 17b) binds poorly to the transiently exposedepitope on gp120 (FIG. 4B), and thus results in only weak neutralizationof fusion or infection.

FIGS. 4D and 4E depict a proposed mechanism of sCD4-SCFV(17b)neutralization of fusion. In the presence of the bispecific chimericfusion protein, the sCD4 domain can bind to gp120 and induce aconformational change in this protein sufficient to permit binding ofthe SCFV(17b) (FIG. 4D). This effectively blocks fusion between the HIVand infection and the target cell.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand.

SEQ ID NO: 1 shows the basic repeat cassette for a linker polypeptide.

SEQ ID NO: 2 shows a seven-repeat polypeptide linker

SEQ ID NO: 3 shows the amino acid sequence of the CD4-SCFv(17b) chimericprotein.

SEQ ID NO: 4 shows the nucleic acid sequence of CD4-SCFv(17b).

SEQ ID NO: 5 shows the pair of synthetic oligonucleotides used to formthe second half of the Stu I site near the 3′ end of CD4 and to producean Spe I overhang at the 3′ end of an intermediate construct (site to bedestroyed upon ligation into pCB-3); the oligonucleotide sequencesreconstruct the remainder of the second domain of CD4 (through ser₁₈₃),and encode an amino acid sequence including ala₁₈₂ser₁₈₃ of CD4 D2 plusan intermediate 37 residue linker (gly₄ser)₆gly₄thr₂ser, followeddirectly by the universal translational termination sequence (UTS).

SEQ ID NO: 6 shows the peptide sequence encoded for by the nucleotidesequences in SEQ ID NO: 5.

SEQ ID NO: 7 shows the forward (5′) primer used to amplify and attachthe 17b SCFv sequence to the CD4-linker sequence in pCD2. Italics showthe region of the primer that overlaps with 17b.

SEQ ID NO: 8 shows the amino acid sequence encoded by theoligonucleotide primer in SEQ ID NO:7. This sequence includes the GlySerresidues at the third (Gly₄Ser) repeat within L1 (encoded by the BamH Isite, followed by the remaining four (Gly₄Ser) repeats, followed by thefirst ten residues of the 17b SCFv (shown in italics).

SEQ ID NO: 9 shows the 3′ primer used to amplify and attach the 17b SCFvsequence plus the thrombin cleavage site and the hexa-his tag to theCD4-linker sequence in pCD2.

SEQ ID NO: 10 shows the peptide encoded for by the nucleotide sequencein SEQ ID NO: 9.

DETAILED DESCRIPTION OF THE INVENTION

I. Abbreviations and Definitions

A. Abbreviations

HIV: human immunodeficiency virus

gp120: the external subunit of the envelope glycoprotein complex of HIV

Env: the envelope glycoprotein complex of HIV

MAb: monoclonal antibody

Fv: antibody “fragment variable”, the variable region of an antibody

SCFv: single-chain antibody variable region

B. Definitions

Unless otherwise noted, technical terms are used according toconventional understanding. Definitions of common terms in molecularbiology may be found in Benjamin Lewin, Genes V, Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc.,1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following definitions of terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Bispecific fusion protein: Proteins that have at least two domains fusedtogether, each domain comprising a binding region capable of forming aspecific complex with a target protein. In general, the two domains aregenetically fused together, in that nucleic acid molecules that encodeeach protein domain are functionally linked together, for instance by alinker oligonucleotide, thereby producing a single fusion-encodingnucleic acid molecule. The translated product of such a fusion-encodingnucleic acid molecule is the bispecific fusion protein.

The two binding regions of such a bispecific protein may associate withtwo different binding determinants or epitopes on a single targetmolecule. One binding domain may bind first to such a target and therebyinduce a conformational change in the target such that the binding ofthe second binding domain to the target is enabled, facilitated, orotherwise increased in affinity. In such an instance, the domain thatbinds first to the target can be referred to as the inducing-bindingdomain, while the domain that binds second is the induced-bindingdomain. These fusion protein domains need not be organized in bindingsequence; the amino-proximal binding domain of the fusion protein may beeither the induced-binding or the inducing-binding domain; likewise forthe carboxy-proximal binding domain.

Bispecific fusion proteins can be further labeled according to thetarget protein they bind to and neutralize. For instance, a bispecificfusion protein according to the current invention that binds to twospecific sites on HIV gp120 protein may be referred to as agp120-targeted bispecific fusion protein.

CD4: Cluster of differentiation factor 4, a T-cell surface protein thatmediates interaction with the MHC class II molecule. CD4 also serves asthe primary receptor site for HIV on T-cells during HIV infection.

Molecules that are derived from CD4 include fragments of CD4, generatedeither by chemical (e.g. enzymatic) digestion or genetic engineeringmeans. Such a fragment may be one or more entire CD4 protein domains(for example, extracellular domains D1, D2, D3, and D4), as defined inthe immunological literature, or a portion of one or more of thesewell-defined domains. For instance, a binding molecule or binding domainderived from CD4 would comprise a sufficient portion of the CD4 proteinto mediate specific and functional interaction between the bindingfragment and a native or viral binding site of CD4. One such bindingfragment includes both the D1 and D2 extracellular domains of CD4 (CD4D1D2), though smaller fragments may also provide specific and functionalCD4-like binding. The gp120-binding site has been mapped to D1 of CD4.

The term “CD4-derived molecules” also encompasses analogs (non-proteinorganic molecules), derivatives (chemically functionalized proteinmolecules obtained starting with the disclosed protein sequences) ormimetics (three-dimensionally similar chemicals) of the native CD4structure, as well as proteins sequence variants or genetic alleles,that maintain the ability to functionally bind to a target molecule.

CD4-induced conformational change: A change induced in thethree-dimensional conformation of the interacting gp120 protein when CD4specifically interacts with gp120 to form a complex. One characteristicof such a change is the exposure of at least one induced epitope on theinteracting gp120 molecule. An epitope induced by such a change iscalled a CD4-induced epitope. Such a CD4-induced epitope may forinstance include gp120 epitopes at or near the co-receptor-bindingregion of the protein.

In addition to CD4 binding, the binding of other molecules may inducethe exposure of induced epitopes on gp120. Such other inducing moleculesare considered CD4-like in terms of their epitope-inducing ability, tothe extent that they expose epitopes congruent with or equivalent tothose induced epitopes exposed upon the binding of native CD4. Theseother inducing molecules include, but in no way are limited to,fragments of CD4, for instance sCD4, or a fragment containing the D1 orD1 and D2 domains of native CD4. A mannose-specific lectin (SC) may alsoserve to expose a CD4-induced epitope (see U.S. Pat. No. 5,843,454), ascan certain anti-gp120 MAbs.

Complex (complexed): Two proteins, or fragments or derivatives thereof,are said to form a complex when they measurably associate with eachother in a specific manner. Such association can be measured in any ofvarious ways, both direct and indirect. Direct methods may includeco-migration in non-denaturing fractionation conditions, for instance.Indirect measurements of association will depend on secondary effectscaused by the association of the two proteins or protein domains. Forinstance, the formation of a complex between a protein and an antibodymay be demonstrated by the antibody-specific inhibition of some functionof the target protein. In the case of gp120, the formation of a complexbetween gp120 and a neutralizing antibody to this protein can bemeasured by determining the degree to which the antibody inhibitsgp120-dependent cell fusion or HIV infectivity. Cell fusion inhibitionand infectivity assays are discussed further below.

Exposing an induced epitope: The process by which two proteins interactspecifically to form a complex (an inducing complex), thereby causing aconformational change in at least one of the two proteins (the targetprotein) such that at least one previously poorly accessible epitope (aninduced epitope) is made accessible to intramolecular interaction. Theformation of such an inducing complex will generally cause the exposureof more than one induced epitope, each of which may be thereby renderedaccessible for intramolecular interaction.

HIV coreceptor: A cell-surface protein other than CD4 involved in theinteraction of HIV virus and its subsequent entry into a target cell.These proteins may also be referred to as fusion coreceptors for HIV.Examples of such coreceptor proteins include, for instance, members ofthe chemokine receptor family (e.g. CXCR4, CCR5, CCR3, and CCR2B).

HIV coreceptor proteins interact with coreceptor binding determinants ofgp120. In general, it is believed that some of these determinants areexposed on gp120 only after the specific interaction of gp120 with CD4,and the consequent CD4-induced conformational change in the interactinggp120. Thus certain HIV coreceptor binding determinants are, or overlapwith, CD4-induced epitopes.

Neutralization of gp120 can be achieved by the specific binding ofneutralizing proteins or protein fragments or domains to one or morecoreceptor binding determinants of gp120, thereby blocking interactionbetween complexed gp120 and the native coreceptor.

HIV neutralizing ability: The measurable ability of a molecule toinhibit infectivity of HIV virus, either in vivo or in vitro. The art isreplete with methods for measuring the neutralizing ability of variousmolecules. Techniques include in vitro peripheral blood mononuclear cell(PBMC) based assays (D'Souza et al., 1997); measurement of virionattachment (Mondor et al., J. Virol. 72:3623–3634, 1998); neutral reddye uptake and antigen capture assays (U.S. Pat. No. 5,695,927);vaccinia-based reporter gene cell fusion assay (Nussbaum et al., J.Virol. 68:5411–5422, 1994) (standard and sCD4 activated assays);productive infection assays (measuring gag antigen p24 or RT synthesis)(Karn, HIV: a practical approach. Oxford Univ. Press, Cambridge, 1995);and infectivity titer reduction assays (Karn, 1995).

In addition, physical interaction between gp120 and CD4 or otherCD4-like molecules can be examined by various methods. See, for instanceU.S. Pat. No. 5,843,454 (measuring conformational changes of gp120 onbinding of various proteins by virus release and susceptibility of gp120to thrombin-mediated cleavage of the V3 loop). Alternately, the abilityof the CD4-like molecule to compete for binding to gp120 with eithernative CD4 or antibody that recognizes the CD4 binding site on gp120(CD4BS) can be measured. This will allow the calculation of relativebinding affinities through standard techniques.

The invention also includes analogs, derivatives or mimetics of thedisclosed fusion proteins, and which have HIV neutralizing ability. Suchmolecules can be screened for HIV neutralizing ability by assaying aprotein similar to the disclosed fusion protein, in that it has one ormore conservative amino acid substitutions, or analogs, derivatives ormimetics thereof, and determining whether the similar protein, analog,derivative or mimetic provides HIV neutralization. The HIVneutralization ability and gp120 binding affinity of these derivativecompounds can be measured by any known means, including those discussedin this application

Injectable composition: A pharmaceutically acceptable fluid compositioncomprising at least one active ingredient, e.g. a bispecific fusionprotein. The active ingredient is usually dissolved or suspended in aphysiologically acceptable carrier, and the composition can additionallycomprise minor amounts of one or more non-toxic auxiliary substances,such as emulsifying agents, preservatives, and pH buffering agents andthe like. Such injectable compositions that are useful for use with thefusion proteins of this invention are conventional; formulations arewell known in the art.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) is one that has been substantiallyseparated or purified away from other biological components in the cellof the organism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Neutralizing antibodies: An antibody that is able to specifically bindto a target protein in such a way as to inhibit the subsequentbiological functioning of that target protein is said to be neutralizingof that biological function. In general, any protein that can performthis type of specific blocking activity is considered a neutralizingprotein; antibodies are therefore a specific class of neutralizingprotein. The complex formed by binding of a neutralizing protein to atarget protein is called a neutralizing complex.

Antibodies that bind to viruses and bacteria and thereby prevent thebinding of these pathogens to target host cells are said to neutralizethe pathogen. Therefore, antibodies that bind to HIV proteins andmeasurably reduce the ability of the virus to bind to or enter targetcells (e.g., T-cells or macrophages) are HIV-neutralizing antibodies. Ingeneral, HIV neutralizing antibodies can be broken down into severaldifferent classes dependent on what region of the viral envelope proteinthe antibody binds to. Broad classes of such antibodies includeanti-gp41 and anti-gp120 antibodies. There are several antigenic regionson the gp120 protein that provide epitopes for the natural or laboratorygeneration of HIV neutralizing antibodies (see WO 98/36087). Broadlycross-reactive neutralizing antibodies usually interact with relativelyinvariant regions of Env.

A primary source of neutralizing antibodies is the peripheral blood ofpatients infected with the HIV virus. Such primary isolates can becloned and/or immortalized using standard techniques. In addition to theisolation of naturally-occurring neutralizing antibodies, proceduresspecifically directed toward their production are known in the art. SeeU.S. Pat. Nos. 5,843,454; 5,695,927; 5,643,756; and 5,013,548 forinstance.

Linker: A peptide, usually between two and 150 amino acid residues inlength that serves to join two protein domains in a multi-domain fusionprotein. Examples of specific linkers can be found, for instance, inHennecke et al. (Protein Eng. 11:405–410, 1998); and U.S. Pat. Nos.5,767,260 and 5,856,456.

Depending on the domains being joined, and their eventual function inthe fusion protein, linkers may be from about two to about 150 aminoacids in length, though these limits are given as general guidance only.The tendency of fusion proteins to form specific and non-specificmultimeric aggregations is influenced by linker length (Alfthan et al.,1998 Protein Eng. 8:725–731, 1998). Thus, shorter linkers will tend topromote multimerization, while longer linkers tend to favor maintenanceof monomeric fusion proteins. Aggregation can also be minimized throughthe use of specific linker sequences, as demonstrated in U.S. Pat. No.5,856,456.

Linkers may be repetitive or non-repetitive. One classical repetitivelinker used in the production of single chain Fvs (SCFvs) is the(Gly₄Ser)₃ (or (GGGGS)₃ or (G₄S)₃) linker. More recently, non-repetitivelinkers have been produced, and methods for the random generation ofsuch linkers are known (Hennecke et al., Protein Eng. 11:405–410, 1998).In addition, linkers may be chosen to have more or less secondarycharacter (e.g. helical character, U.S. Pat. No. 5,637,481) depending onthe conformation desired in the final fusion protein. The more secondarycharacter a linker possesses, the more constrained the structure of thefinal fusion protein will be. Therefore, substantially flexible linkersthat are substantially lacking in secondary structure allow flexion ofthe fusion protein at the linker.

A linker is capable of retaining a binding domain of a protein inbinding proximity of a target binding site when the linker is ofsufficient length and flexibility to allow specific interaction betweenthe binding domain and the target binding site. In the case of thebispecific fusion proteins of this invention, a linker that maintainsbinding proximity permits the sequential binding with the target offirst the inducing-binding domain of the fusion protein, then theinduced-binding domain. A linker that maintains the domains of abispecific fusion protein in binding proximity to a target can beconsidered an operable or functional linker as relates to such abispecific fusion protein.

Oligonucleotide: A linear polynucleotide sequence of between six and 300nucleotide bases in length.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any internal termination codons. Thesesequences are usually translatable into a peptide.

Parenteral: Administered outside of the intestine, e.g., not via thealimentary tract. Generally, parenteral formulations are those that willbe administered through any possible mode except ingestion. This termespecially refers to injections, whether administered intravenously,intrathecally, intramuscularly, intraperitoneally, or subcutaneously,and various surface applications including intranasal, intradermal, andtopical application, for instance.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified fusionprotein preparation is one in which the fusion protein is more enrichedthan the protein is in its generative environment, for instance within acell or in a biochemical reaction chamber. In some embodiments, apreparation of fusion protein is purified such that the fusion proteinrepresents at least 50% of the total protein content of the preparation.

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring or has a sequence that is madeby an artificial combination of two otherwise separated segments ofsequence. This artificial combination can be accomplished by chemicalsynthesis or, more commonly, by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques.

Similarly, a recombinant protein is one encoded for by a recombinantnucleic acid molecule.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences is expressed in terms of the similarity betweenthe sequences, otherwise referred to as sequence identity. Sequenceidentity is frequently measured in terms of percentage identity (orsimilarity or homology); the higher the percentage, the more similar thetwo sequences are. Homologs of the bispecific fusion protein willpossess a relatively high degree of sequence identity when aligned usingstandard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman (Adv. Appl. Math. 2: 482, 1981); Needleman and Wunsch (J.Mol. Biol. 48: 443–453, 1970); Pearson and Lipman (Proc. Natl. Acad.Sci., USA 85:2444–2448, 1988); Higgins and Sharp (Gene, 73:237–244,1988); Higgins and Sharp (CABIOS 5:151–153, 1989); Corpet et al. (Nuc.Acids Res. 16: 10881–10890, 1988); Huang et al. (Comp. Appls. Biosci.8:155–165, 1992); and Pearson et al. (Methods in Molecular Biology 24:307–331, 1994). Altschul et al. (Nature Genet., 6:119–129, 1994)presents a detailed consideration of sequence alignment methods andhomology calculations.

The alignment tools ALIGN (Myers and Miller, CABIOS 4:11–17, 1989) orLFASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., USA 85:2444–2448,1988) may be used to perform sequence comparisons (Internet Program©1996, W. R. Pearson and the University of Virginia, “fasta20u63” version2.0u63, release date December 1996). ALIGN compares entire sequencesagainst one another, while LFASTA compares regions of local similarity.These alignment tools and their respective tutorials are available onthe Internet.

Orthologs of the disclosed bispecific fusion proteins are typicallycharacterized by possession of greater than 75% sequence identitycounted over the full-length alignment with the amino acid sequence ofbispecific fusion protein using ALIGN set to default parameters.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., JMol. Biol. 1990 215:403–410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI BLAST website. A description of how todetermine sequence identity using this program is also available at theNCBI website BLAST tutorial.

For comparisons of amino acid sequences of greater than about 30 aminoacids, the “Blast 2 sequences” function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least90%, at least 92%, at least 94%, at least 95%, at least 97%, at least98%, or at least 99% sequence identity. In addition, sequence identitycan be compared over the full length of one or both binding domains ofthe disclosed fusion proteins. In such an instance, percentageidentities will be essentially similar to those discussed forfull-length sequence identity.

When significantly less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 80% sequenceidentity over short windows of 10–20 amino acids, and may possesssequence identities of at least 85%, at least 90%, at least 95%, or atleast 99% depending on their similarity to the reference sequence.Sequence identity over such short windows can be determined usingLFASTA; methods are described on the Internet. One of skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided. Thepresent invention provides not only the peptide homologs that aredescribed above, but also nucleic acid molecules that encode suchhomologs.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Conditions for nucleic acid hybridization andcalculation of stringencies can be found in Sambrook et al. (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989)and Tijssen (Laboratory Techniques in Biochemistry and Molecular BiologyPart I, Ch. 2, Elsevier, N.Y., 1993). Nucleic acid molecules thathybridize under stringent conditions to the disclosed bispecific fusionprotein sequences will typically hybridize to a probe based on eitherthe entire fusion protein encoding sequence, an entire binding domain,or other selected portions of the encoding sequence under washconditions of 0.2×SSC, 0.1% SDS at 65° C.

Nucleic acid sequences that do not show a high degree of identity maynevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid sequences, each encoding substantially the same protein.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a gp120-specific binding agent binds substantiallyonly the gp120 protein. As used herein, the term “gp120-specific bindingagent” includes anti-gp120 antibodies and other agents that bindsubstantially only to a gp120 protein.

Anti-gp120 antibodies may be produced using standard proceduresdescribed in a number of texts, including Harlow and Lane (UsingAntibodies, A Laboratory Manual, CSHL, New York, 1999, ISBN0-87969-544-7). In addition, certain techniques may enhance theproduction of neutralizing antibodies (U.S. Pat. Nos. 5,843,454;5,695,927; 5,643,756; and 5,013,548). The determination that aparticular agent binds substantially only to gp120 protein may readilybe made by using or adapting routine procedures. One suitable in vitroassay makes use of the Western blotting procedure (described in manystandard texts, including Harlow and Lane, 1999). Western blotting maybe used to determine that a given protein binding agent, such as ananti-gp120 monoclonal antibody, binds substantially only to the MSGprotein. Antibodies to gp120 are well known in the art.

Shorter fragments of antibodies can also serve as specific bindingagents. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) that bindto gp120 would be gp120-specific binding agents.

Therapeutically effective amount of a bispecific fusion protein: Aquantity of bispecific fusion protein sufficient to achieve a desiredeffect in a subject being treated. For instance, this can be the amountnecessary to inhibit viral proliferation or to measurably neutralizedisease organism binding mechanisms. In general, this amount will besufficient to measurably inhibit virus (e.g. HIV) replication orinfectivity.

An effective amount of bispecific fusion protein may be administered ina single dose, or in several doses, for example daily, during a courseof treatment. However, the effective amount of fusion protein will bedependent on the fusion protein applied, the subject being treated, theseverity and type of the affliction, and the manner of administration ofthe fusion protein. For example, a therapeutically effective amount offusion protein can vary from about 0.01 mg/kg body weight to about 1g/kg body weight.

The fusion proteins disclosed in the present invention have equalapplication in medical and veterinary settings. Therefore, the generalterm “subject being treated” is understood to include all animals (e.g.humans, apes, dogs, cats, horses, and cows) that are or may be infectedwith a virus or other disease-causing microorganism that is susceptibleto bispecific fusion protein-mediated neutralization.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

II. Construction, Expression, and Purification of Bispecific FusionProteins.

A. Selection of Component Domains.

This invention provides generally a bispecific fusion protein that bindsto two different sites on a target protein. As such, any target proteinthat has two different binding sites is an example of a target for abispecific fusion protein. Particular targets include proteins on whichone of the two binding sites (the induced-binding site) isexposed/induced by the binding of the fusion protein to a first bindingsite (the inducing-binding site) on the target. The choice of proteinbinding domains for incorporation into the disclosed bispecific fusionprotein will be dictated by the target protein chosen. The choice oflinker will also be influenced by the target protein and binding siteschosen. In general, the linker used in any bispecific fusion will be ofa length and secondary character to hold the induced-binding domainwithin binding proximity of the target protein induced binding site,once the inducing-binding domain of the fusion protein has formed aspecific complex with the target.

In certain embodiments, the target protein is an HIV envelopeglycoprotein, for instance HIV-1 gp120. In certain of these and otherembodiments, the inducing-binding site is the CD4 binding site on gp120.As such, the inducing-binding domain of the disclosed bispecific fusionprotein can be a binding fragment of CD4, for instance sCD4.Alternately, any other molecule that specifically interacts with gp120in such a way as to expose one or more induced epitopes would also serveas the source of an inducing-binding protein domain. The specificfragments used to construct the fusion protein should be chosen so thatthe conformation of the final fusion provides functional and inducingbinding to gp120; this can be assayed either directly (e.g., affinitymeasurements) or indirectly (e.g., neutralization assays).

Non-CD4-derived CD4 mimics may also be employed as sources forinducing-binding domains of the disclosed fusion proteins. For instance,a mannose-specific lectin (SC) may serve to induce CD4 inducedconformational changes (see U.S. Pat. No. 5,843,454). Alternatively,antibodies that bind the CD4-binding site or another epitope of gp120and thereby induce a CD4-like conformational change on the complexedprotein can also be used.

Non-peptide CD4 analogs can also be used in this invention, for instanceorganic or non-organic structural analog of the gp120-interactingdomain(s) of the CD4 molecule.

Induced-binding domains of a gp120-targeted fusion protein will includeantibodies (or fragments thereof) that recognize induced epitopes of thecomplexed gp120. In some embodiments, such antibodies are broadlycross-reactive against diverse HIV-1 isolates. Induced epitopes includeall of those referred to as CD4-induced (CD4i) epitopes, and inparticular those which overlap coreceptor-binding determinants of gp120.Previously identified neutralizing monoclonal antibodies can be used,and include but are not limited to human monoclonal antibodies 17b, 48d,and CG10.

Likewise, induced binding domains of the disclosed chimeric moleculescan be non-peptide molecules, for instance organic or non-organicstructural analogs of SCFv(17b).

In addition to antibodies that bind induced epitopes of gp120, othersources for induced-binding domains include fragments of coreceptorsthat specifically interact with a coreceptor binding domain(s) of gp120.

The construction of a gp120-specific bispecific fusion protein can beaided by review of the X-ray crystallographic structure of the ternarycomplex containing the gp120 core, a two-domain fragment of CD4 (D1D2),and an FAb from a broadly cross-reactive human MAb (17b) directedagainst the coreceptor-binding determinants of gp120 (Kwong et al.,Nature 393:648–659, 1998). Computer-based examination of the structuralcoordinates of this ternary complex, using FRODO (Jones et al, Meth.Enzymol. 115:157–171, 1985; Jones, J. Appl. Cryst. 11:268–272, 1978;Pflugrath et al. Methods and Applications in Crystallography, pages407–420, Clarendon Press, Oxford), has revealed choices for constructingthe chimeric protein. The shortest distance between free termini of CD4and the 17b FAb is 56 Å, i.e. from the free C-terminus of the D1D2 sCD4fragment to the N-terminus of the 17b FAb heavy chain. A linkerconnecting these termini would be essentially free of steric hindrancefrom CD4 and the N-terminus of the 17b light chain. Possible connectionscould also be made between the N-terminus of CD4 and the C-termini ofthe 17b heavy or light chains; such connections would require linkers ofabout 65 Å and about 86 Å, respectively. In the latter two connectionsthe linker is required to circumvent other portions of the complex,including the bulky variable loops.

B. Assembly.

The construction of chimeric molecules, in particular fusion proteins,from domains of known proteins is well known. In general, a nucleic acidmolecule that encodes the desired protein domains are joined usingstandard genetic engineering techniques to create a single, operablylinked fusion oligonucleotide. Molecular biological techniques may befound in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., 1989). Specific examples of geneticallyengineered multi-domain proteins, especially those based on molecules ofthe immunoglobulin superfamily, joined by various linkers, can be foundin the following patent documents:

U.S. Pat. No. 5,856,456 (“Linker for linked fusion polypeptides”);

U.S. Pat. No. 5,696,237 (“Recombinant antibody-toxin fusion protein”);

U.S. Pat. No. 5,767,260 (“Antigen-binding fusion proteins”);

U.S. Pat. No. 5,587,455 (“Cytotoxic agent against specific virusinfection”); and

WO 98/36087 (“Immunological tolerance to HIV epitopes”).

Non-peptide analogs that serve as inducing-binding or induced bindingdomains of the invention can be linked to the opposite domain of thechimeric molecules using known chemical linking techniques, includingchemical cross-linking. Cross-linkers are well known, and examples ofmolecules used for cross-linking can be found, for instance, in U.S.Pat. No. 6,027,890 (“Methods and compositions for enhancing sensitivityin the analysis of biological-based assays”).

C. Expression.

One skilled in the art will understand that there are myriad ways toexpress a recombinant protein such that it can subsequently be purified.In general, an expression vector carrying the nucleic acid sequence thatencodes the desired protein will be transformed into a microorganism forexpression. Such microorganisms can be prokaryotic (bacteria) oreukaryotic (e.g., yeast). One example species of bacteria that can beused is Escherichia coli (E. coli), which has been used extensively as alaboratory experimental expression system. An eukaryotic expressionsystem can be used where the protein of interest requireseukaryote-specific post-translational modifications such asglycosylation. Also, protein can be expressed using a viral (e.g.,vaccinia) based expression system.

Protein can also be expressed in animal cell tissue culture, and such asystem can be used where animal-specific protein modifications aredesirable or required in the recombinant protein.

The expression vector can include a sequence encoding a targetingpeptide, positioned in such a way as to be fused to the coding sequenceof the bispecific fusion protein. This allows the bispecific fusionprotein to be targeted to specific sub-cellular or extra-cellularlocations. Various prokaryotic and eukaryotic targeting peptides, andnucleic acid molecules encoding such, are known. In a prokaryoticexpression system, a signal sequence can be used to secrete the newlysynthesized protein. In an eukaryotic expression system, the targetingpeptide would specify targeting of the hybrid protein to one or morespecific sub-cellular compartments, or to be secreted from the cell,depending on which peptide is chosen. Through the use of an eukaryoticsecretion signal sequence, the bispecific fusion protein can beexpressed in a transgenic animal (for instance a cow, pig, or sheep) insuch a manner that the protein is secreted into the milk of the animal.

Vectors suitable for stable transformation of culturable cells are alsowell known. Typically, such vectors include a multiple-cloning sitesuitable for inserting a cloned nucleic acid molecule, such that it willbe under the transcriptional control of 5′ and 3′ regulatory sequences.In addition, transformation vectors include one or more selectablemarkers; for bacterial transformation this is often an antibioticresistance gene. Such transformation vectors typically also contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, and atranscription termination site, each functionally arranged in relationto the multiple-cloning site. For production of large amounts ofrecombinant proteins, an inducible promoter can be used. This permitsselective production of the recombinant protein, and allows both higherlevels of production than constitutive promoters, and enables theproduction of recombinant proteins that may be toxic to the expressingcell if expressed constitutively.

In addition to these general guidelines, protein expression/purificationkits are produced commercially. See, for instance, the QIAEXPRESS™expression system from QIAGEN (Chatsworth, Calif.) and variousexpression systems provided by INVITROGEN (Carlsbad, Calif.). Dependingon the details provided by the manufactures, such kits can be used forproduction and purification of the disclosed bispecific fusion proteins.

D. Purification.

One skilled in the art will understand that there are myriad ways topurify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify the disclosed bispecific fusionproteins. Such methods include, for instance, protein chromatographicmethods including ion exchange, gel filtration, HPLC, monoclonalantibody affinity chromatography and isolation of insoluble proteininclusion bodies after over production. In addition, purificationaffinity-tags, for instance a six-histidine sequence, may berecombinantly fused to the protein and used to facilitate polypeptidepurification. A specific proteolytic site, for instance athrombin-specific digestion site, can be engineered into the proteinbetween the tag and the fusion itself to facilitate removal of the tagafter purification.

Commercially produced protein expression/purification kits providetailored protocols for the purification of proteins made using eachsystem. See, for instance, the QIAEXPRESS™ expression system from QIAGEN(Chatsworth, Calif.) and various expression systems provided byINVITROGEN (Carlsbad, Calif.). Where a commercial kit is employed toproduce a bispecific fusion protein, the manufacturer's purificationprotocol is a particularly disclosed protocol for purification of thatprotein. For instance, proteins expressed with an amino-terminalhexa-his tag can be purified by binding to nickel-nitrilotriacetic acid(Ni-NTA) metal affinity chromatography matrix (The QIAexpressionist,QIAGEN, 1997).

Alternately, the binding specificities of either the first or secondbinding domains, or both, of the disclosed fusion protein may beexploited to facilitate specific purification of the proteins. Onemethod of performing such specific purification would be columnchromatography using column resin to which the target molecule, or anepitope or fragment or domain of the target molecule, has been attached.

If the bispecific fusion protein is produced in a secreted form, e.g.secreted into the milk of a transgenic animal, purification will be fromthe secreted fluid. Alternately, purification may be unnecessary if thefusion protein can be applied directly to the subject in the secretedfluid (e.g. milk).

III. Variation of a Bispecific Fusion Protein

A. Sequence Variants

The binding characteristics and therefore neutralizing activity of thefusion proteins disclosed herein lies not in the precise amino acidsequence, but rather in the three-dimensional structure inherent in theamino acid sequences encoded by the DNA sequences. It is possible torecreate the binding characteristics of any of these proteins or proteindomains of this invention by recreating the three-dimensional structure,without necessarily recreating the exact amino acid sequence. This canbe achieved by designing a nucleic acid sequence that encodes for thethree-dimensional structure, but which differs, for instance by reasonof the redundancy of the genetic code. Similarly, the DNA sequence mayalso be varied, while still producing a functional neutralizing protein.

Variant neutralizing bispecific binding proteins include proteins thatdiffer in amino acid sequence from the disclosed sequence, but thatshare structurally significant sequence homology with any of theprovided proteins. Variation can occur in any single domain of thefusion protein (e.g. the first or second binding domain, or the linker).Variation can also occur in more than one of such domains in anyparticular variant protein. Such variants may be produced bymanipulating the nucleotide sequence of the, for instance aCD4-SCFv(17b)-encoding sequence, using standard procedures, includingsite-directed mutagenesis or PCR. The simplest modifications involve thesubstitution of one or more amino acids for amino acids having similarbiochemical properties. These so-called conservative substitutions arelikely to have minimal impact on the activity of the resultant protein,especially when made outside of the binding site of each domain. Table 1shows amino acids that may be substituted for an original amino acid ina protein, and which are regarded as conservative substitutions.

TABLE 1 Original Residue Conservative Substitutions Ala ser Arg lys Asngln; his Asp glu Cys ser Gln asn Glu asp Gly pro His asn; gln Ile leu;val Leu ile; val Lys arg; gln; glu Met leu; ile Phe met; leu; tyr Serthr Thr ser Trp tyr Tyr trp; phe Val ile; leu

More substantial changes in protein structure may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 1. Such changes include changing residues that differmore significantly in their effect on maintaining polypeptide backbonestructure (e.g., sheet or helical conformation) near the substitution,charge or hydrophobicity of the molecule at the target site, or bulk ofa specific side chain. The following substitutions are generallyexpected to produce the greatest changes in protein properties: (a) ahydrophilic residue (e.g., seryl or threonyl) is substituted for (or by)a hydrophobic residue (e.g., leucyl, isoleucyl, phenylalanyl, valyl oralanyl); (b) a cysteine or proline is substituted for (or by) any otherresidue; (c) a residue having an electropositive side chain (e.g.,lysyl, arginyl, or histadyl) is substituted for (or by) anelectronegative residue (e.g., glutamyl or aspartyl); or (d) a residuehaving a bulky side chain (e.g., phenylalanine) is substituted for (orby) one lacking a side chain (e.g., glycine).

Variant binding domain or fusion protein-encoding sequences may beproduced by standard DNA mutagenesis techniques, for example, M13 primermutagenesis. Details of these techniques are provided in Sambrook (InMolecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989),Ch. 15. By the use of such techniques, variants may be created whichdiffer in minor ways from the bispecific fusion protein-encodingsequences disclosed. DNA molecules and nucleotide sequences which arederivatives of those specifically disclosed herein and that differ fromthose disclosed by the deletion, addition, or substitution ofnucleotides while still encoding a protein that binds twice to gp120,thereby neutralizing HIV virus infectivity, are comprehended by thisinvention. In their most simple form, such variants may differ from thedisclosed sequences by alteration of the coding region to fit the codonusage bias of the particular organism into which the molecule is to beintroduced.

Alternatively, the coding region may be altered by taking advantage ofthe degeneracy of the genetic code to alter the coding sequence suchthat, while the nucleotide sequence is substantially altered, itnevertheless encodes a protein having an amino acid sequencesubstantially similar to the disclosed fusion sequences. For example,the 18th amino acid residue of the CD4-SCFv(17b) protein (after cleavageof the N-terminal signal sequence) is alanine. The nucleotide codontriplet GCT encodes this alanine residue. Because of the degeneracy ofthe genetic code, three other nucleotide codon triplets—(GCG, GCC andGCA)—also code for alanine. Thus, the nucleotide sequence of thedisclosed CD4-SCFv(17b) encoding sequence could be changed at thisposition to any of these three alternative codons without affecting theamino acid composition or characteristics of the encoded protein. Basedupon the degeneracy of the genetic code, variant DNA molecules may bederived from the cDNA and gene sequences disclosed herein using standardDNA mutagenesis techniques as described above, or by synthesis of DNAsequences. Thus, this invention also encompasses nucleic acid sequenceswhich encode a neutralizing bispecific fusion protein, but which varyfrom the disclosed nucleic acid sequences by virtue of the degeneracy ofthe genetic code.

B. Peptide Modifications

The present invention includes biologically active molecules that mimicthe action of the bispecific fusion proteins of the present invention,and specifically neutralize HIV Env. The proteins of the inventioninclude synthetic embodiments of naturally-occurring proteins describedherein, as well as analogues (non-peptide organic molecules),derivatives (chemically functionalized protein molecules obtainedstarting with the disclosed peptide sequences) and variants (homologs)of these proteins that specifically bind with and neutralize HIV gp120.Each protein of the invention is comprised of a sequence of amino acids,which may be either L- and/or D-amino acids, naturally occurring andotherwise.

Proteins may be modified by a variety of chemical techniques to producederivatives having essentially the same activity as the unmodifiedproteins, and optionally having other desirable properties. For example,carboxylic acid groups of the protein, whether carboxyl-terminal or sidechain, may be provided in the form of a salt of apharmaceutically-acceptable cation or esterified to form a C₁–C₁₆ ester,or converted to an amide of formula NR₁R₂ wherein R₁ and R₂ are eachindependently H or C₁–C₁₆ alkyl, or combined to form a heterocyclicring, such as a 5- or 6-membered ring. Amino groups of the protein,whether amino-terminal or side chain, may be in the form of apharmaceutically-acceptable acid addition salt, such as the HCl, HBr,acetic, benzoic, toluene sulfonic, maleic, tartaric and other organicsalts, or may be modified to C₁–C₁₆ alkyl or dialkyl amino or furtherconverted to an amide.

Hydroxyl groups of the protein side chains may be converted to C₁–C₁₆alkoxy or to a C₁–C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the protein side chains may be substituted with one ormore halogen atoms, such as fluorine, chlorine, bromine or iodine, orwith C₁–C₁₆ alkyl, C₁–C₁₆ alkoxy, carboxylic acids and esters thereof,or amides of such carboxylic acids. Methylene groups of the protein sidechains can be extended to homologous C₂–C₄ alkylenes. Thiols can beprotected with any one of a number of well-recognized protecting groups,such as acetamide groups. Those skilled in the art will also recognizemethods for introducing cyclic structures into the proteins of thisinvention to select and provide conformational constraints to thestructure that result in enhanced stability.

It also may be advantageous to introduce one or more disulfide bonds toconnect the frameworks of the heavy and light chains in the SCFv domain.This modification often enhances the stability and affinity of SCFvs(Reiter et al., Protein Engineering 7:697–704, 1994). Here too, theX-ray crystal structure containing the 17 FAb (Kwong et al., Nature393:648–659, 1998) can be used to assess optimal sites for engineeringcysteine residues of the heavy and light chains.

Peptidomimetic and organomimetic embodiments are also within the scopeof the present invention, whereby the three-dimensional arrangement ofthe chemical constituents of such peptido- and organomimetics mimic thethree-dimensional arrangement of the protein backbone and componentamino acid side chains in the bispecific neutralizing fusion protein,resulting in such peptido- and organomimetics of the proteins of thisinvention having measurable or enhanced neutralizing ability. Forcomputer modeling applications, a pharmacophore is an idealized,three-dimensional definition of the structural requirements forbiological activity. Peptido- and organomimetics can be designed to fiteach pharmacophore with current computer modeling software (usingcomputer assisted drug design or CADD). See Walters, “Computer-AssistedModeling of Drugs”, in Klegerman & Groves, eds., 1993, PharmaceuticalBiotechnology, Interpharm Press: Buffalo Grove, Ill., pp. 165–174 andPrinciples of Pharmacology Munson (ed.) 1995, Ch. 102, for descriptionsof techniques used in CADD. Also included within the scope of theinvention are mimetics prepared using such techniques that produceneutralizing fusion proteins.

C. Domain Length Variation.

It will be appreciated that the protein domains of the current inventionmay be combined to produce fusion protein molecules without necessarilysplicing the components in the same place. It is believed to be possibleto use shorter or longer fragments of each component domain, linked by afunctional linker. For instance, any component which is spliced withinabout 10 amino acid residues of the residue specified, and which stillprovides a functional binding fragment, comprises about the same domain.However, domains of substantially longer or substantially shorter lengthcan be used. For instance, in certain embodiments, the protein caninclude a leader sequence plus a four-domain CD4 (D1–D4, amino acidresidues 1–372), or just the first domain of CD4 (D1 residues 1–113).

IV. Activity of Fusion Proteins

It is important to assess the chemical, physical and biological activityof the disclosed bispecific fusion proteins. Among other uses, suchassays permit optimization of the domains chosen, as well asoptimization of the length and conformation of the linkers used toconnect them. Control molecules should be included in each assay;usually such will include each domain alone, as well as the two domainsas separate molecules mixed in the reaction, for instance in a 1:1 molarratio. In the case of a CD4-SCFv(17b) bispecific fusion protein, suchcontrols would include sCD4 and SCFv(17b), for instance.

A. Fusion Protein Affinity for Target Protein

Fusion protein affinity for the target protein can be determined usingvarious techniques. For instance, co-immunoprecipitation analyses withmetabolically labeled proteins can be employed to determine binding ofsCD4-SCFv proteins, e.g. sCD4-SCFv(17b) to soluble HIV-1 gp120, usinganti-gp120 MAbs that do not interfere with CD4 binding (e.g. MAb D47that binds to V3), or polyclonal antibody to the C-terminus of gp120.ELISA can also be used to examine the binding characteristics of eachdomain of the chimera.

B. Neutralization Assays

Various assays can be used to measure the ability of the disclosedfusion proteins to inhibit function of the target protein. Individualcomponents of the fusion protein will serve as controls. In general,assays will be specific for the target/fusion protein. For instance,many functional analyses can test the ability of sCD4-SCFv fusions toneutralize the HIV Env. It is particularly advantageous to use Envs fromdiverse HIV-1 strains to test the breadth of inhibition (neutralizingability) of each fusion protein for different HIV-1 genetic subtypes anddifferent phenotypes (i.e. coreceptor usage). In addition, it isadvantageous to test such fusion proteins in the standard andsCD4-activated assays for Env-mediated cell fusion. Known HIV-1neutralizing MAbs and MAbs against CD4-induced epitopes on gp120 areexamples of controls for such experiments. Possible synergisticinhibition with other known broadly cross-reactive neutralizing MAbsshould be tested (e.g. bl2, 2F5, F105, 2G12).

In the case of gp120-targeted fusion proteins, the vaccinia-basedreporter gene cell fusion assay may be used to assess fusion inhibition(Nussbaum et al., J. Virol. 68:5411–5422, 1994). One population oftissue culture cells (e.g. BS-C-1, HeLa, or NIH 3T3) uniformlyexpressing vaccinia virus-encoded binding and fusion-mediating viralenvelope glycoprotein(s) is mixed with another population expressing thecorresponding cellular receptor(s). In the case of sCD4-SCFv fusions,where the target protein is HIV-1 gp120, one cell population expressesHIV-1 Env, while the other expresses necessary HIV-1 receptors (e.g. CD4and a chemokine receptor). The cytoplasm of either cell population alsocontains vaccinia virus-encoded bacteriophage T7 DNA polymerase; thecytoplasm of the other contains a transfected plasmid with the E. colilacZ gene linked to the T7 promoter. Upon mixing of the two populations,cell fusion results in activation of the lacZ gene, through theintroduction of the T7 RNA polymerase into proximity with thetransfected T7 promoter-lacZ in the cytoplasm of the fused cells. Theresultant β-galactosidase (β-gal) activity is proportional to the amountof fusion that occurs, and can be measured by colorimetric assay ofdetergent cell lysates or in situ staining. Cell-fusion neutralizingactivity of bispecific fusion proteins is therefore assessed bymeasuring their inhibition of β-gal production.

The gp120-targeted fusions (e.g. sCD4-SCFv) can also be tested forability to block HIV-1 infection using single round assays (e.g. usingindicator cell lines, Vodicka et al., Virology 233:193–198, 1997).Target cells expressing CD4 and a specific coreceptor, and containingthe lacZ reporter gene linked to the HIV-1 long terminal repeat (LTR),are infected with specific HIV-1 strains (Vodicka, 1997). Integration ofan HIV provirus in these cells leads to production of the viraltransactivator, Tat, which then turns on expression of the B-gal genevia interaction with LTR. The activity of sCD4-SCFv is assessed by itsinhibition of production of β-gal-positive cells (stained blue withX-gal), which is proportional to its ability to block HIV-1 infection.

V. Incorporation of Bispecific Fusion Proteins into PharmaceuticalCompositions

Pharmaceutical compositions that comprise at least one bispecific fusionprotein as described herein as an active ingredient will normally beformulated with a solid or liquid carrier, depending upon the particularmode of administration chosen. The pharmaceutically acceptable carriersand excipients useful in this invention are conventional. For instance,parenteral formulations usually comprise injectable fluids that arepharmaceutically and physiologically acceptable fluid vehicles such aswater, physiological saline, other balanced salt solutions, aqueousdextrose, glycerol or the like. Excipients that can be included are, forinstance, other proteins, such as human serum albumin or plasmapreparations. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Other medicinal and pharmaceutical agents, for instance nucleosidederivatives (e.g. AZT) or protease inhibitors, also may be included. Itmay also be advantageous to include other fusion inhibitors, forinstance one or more neutralizing antibodies.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical and oral formulations can be employed.Topical preparations can include eye drops, ointments, sprays and thelike. Oral formulations may be liquid (e.g., syrups, solutions orsuspensions), or solid (e.g., powders, pills, tablets, or capsules). Forsolid compositions, conventional non-toxic solid carriers can includepharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that comprise bispecific fusion proteinmay be formulated in unit dosage form, suitable for individualadministration of precise dosages. One possible unit dosage containsapproximately 100 μg of protein. The amount of active compoundadministered will be dependent on the subject being treated, theseverity of the affliction, and the manner of administration, and isbest left to the judgment of the prescribing clinician. Within thesebounds, the formulation to be administered will contain a quantity ofthe active component(s) in an amount effective to achieve the desiredeffect in the subject being treated.

VI. Clinical Use of Bispecific Fusion Proteins

The potent viral-neutralizing activity exhibited by the disclosedbispecific fusion proteins makes them useful for treating viralinfections in human and other animal subjects. Possibly susceptibleviruses include the immunodeficiency viruses, such as HIV and similar orrelated viruses in simians and other animals. In addition, other viralor microbial systems that involve the interaction of a first inducingand second induced binding site of a single protein will also besusceptible to neutralization using bispecific fusion proteins of thecurrent invention. The bispecific fusion proteins disclosed herein canalso be used in highly sensitive detection or purification of targetprotein.

The bispecific fusion proteins of this invention may be administered tohumans, or other animals on whose cells they are effective, in variousmanners such as topically, orally, intravenously, intramuscularly,intraperitoneally, intranasally, intradermally, intrathecally, andsubcutaneously. The particular mode of administration and the dosageregimen will be selected by the attending clinician, taking into accountthe particulars of the case (e.g., the subject, the disease, and thedisease state involved, and whether the treatment is prophylactic orpost-infection). Treatment may involve daily or multi-daily doses ofbispecific fusion protein(s) over a period of a few days to months, oreven years.

If treatment is through the direct administration of cells expressingthe bispecific fusion protein to the subject, such cells (e.g.transgenic pluripotent or hematopoietic stem cells or B cells) may beadministered at a dose of between about 10⁶ and 10¹⁰ cells, on one orseveral occasions. The number of cells will depend on the patient, aswell as the fusion protein and cells chosen to express the protein.

A general strategy for transferring genes into donor cells is disclosedin U.S. Pat. No. 5,529,774, which is incorporated by reference.Generally, a gene encoding a protein having therapeutically desiredeffects is cloned into a viral expression vector, and that vector isthen introduced into the target organism. The virus infects the cells,and produces the protein sequence in vivo, where it has its desiredtherapeutic effect. See, for example, Zabner et al., Cell 75:207–216,1993. As an alternative to adding the sequences encoding the bispecificfusion protein or a homologous protein to the DNA of a virus, it is alsopossible to introduce such a gene into the somatic DNA of infected oruninfected cells, by methods that are well known in the art (Sambrook etal., In Molecular Cloning: A Laboratory Manual, Cold Spring Harbor,N.Y., 1989). These methods can be used to introduce the herein disclosedfusion proteins to human cells to provide long-term resistance to HIV-1infection or AIDS. For example, gene therapy can be used to secrete theprotein at mucosal surfaces, such as the vaginal, rectal, or oralmucosa.

HIV-1 gp120-targeted bispecific fusion proteins, for instancesCD4-SCFv(17b), are particularly useful in the prevention of infectionduring or immediately after HIV exposure (e.g., mother/infanttransmission, post-exposure prophylaxis, and as a topical inhibitor). Insuch instances, one or more doses of the bispecific fusion protein areadministered before or soon after the triggering event. To prevent orameliorate mother/infant transmission of viral infection, for instance,it may be beneficial to administer the gp120-targeted bispecific fusionprotein to the mother intermittently throughout pregnancy, and/orimmediately before or following delivery, and/or directly to the newbornimmediately after birth. Post-exposure prophylactic treatments may beparticularly beneficial where there has been accidental exposure (forinstance, a medically related accidental exposure), including but notlimited to a contaminated needle-stick or medical exposure to HIV-1contaminated blood or other fluid.

The present invention also includes combinations of chimeric bispecificfusion proteins with one or more other agents useful in the treatment ofdisease, e.g. HIV disease. For example, the compounds of this inventionmay be administered, whether before or after exposure to the virus, incombination with effective doses of other anti-virals, immunomodulators,anti-infectives, and/or vaccines. The term “administration incombination” refers to both concurrent and sequential administration ofthe active agents.

Examples of antiviral agents that can be used in combination with thechimeric bispecific fusion proteins of the invention are: AL-721 (fromEthigen of Los Angeles, Calif.), recombinant human interferon beta (fromTriton Biosciences of Alameda, Calif.), Acemannan (from Carrington Labsof Irving, Tex.), gangiclovir (from Syntex of Palo alto, Calif.),didehydrodeoxythymidine or d4T (from Bristol-Myers-Squibb), EL10 (fromElan Corp. of Gainesville, Ga.), dideoxycytidine or ddC (fromHoffman-LaRoche), Novapren (from Novaferon labs, Inc. of Akron, Ohio),zidovudine or AZT (from Burroughs Wellcome), ribaririn (from Viratek ofCosta Mesa, Calif.), alpha interferon and acyclovir (from BurroughsWellcome), Indinavir (from Merck & Co.), 3TC (from Glaxo Wellcome),Ritonavir (from Abbott), Saquinavir (from Hoffman-LaRoche), and others.

Examples of immuno-modulators that can be used in combination with thechimeric bispecific fusion proteins of the invention are AS101(Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon(Genentech), GM-CSF (Genetics Institute), IL-2 (Cetus orHoffman-LaRoche), human immune globulin (Cutter Biological), IMREG (fromImreg of New Orleans, La.), SK&F106528, and TNF (Genentech).

Examples of some anti-infectives with which the chimeric bispecificfusion proteins can be used include clindamycin with primaquine (fromUpjohn, for the treatment of Pneumocystis pneumonia), fluconazlone (fromPfizer for the treatment of cryptococcal meningitis or candidiasis),nystatin, pentamidine, trimethaprim-sulfamethoxazole, and many others.

The combination therapies are of course not limited to the listsprovided in these examples, but includes any composition for thetreatment of HIV disease (including treatment of AIDS).

VII. Kits

The chimeric proteins disclosed herein can be supplied in the form of akit for use in prevention and/or treatment of diseases (e.g., HIVinfection and AIDS). In such a kit, a clinically effective amount of oneor more of the chimeric bispecific fusion proteins is provided in one ormore containers. The chimeric bispecific fusion proteins may be providedsuspended in an aqueous solution or as a freeze-dried or lyophilizedpowder, for instance. In certain embodiments, the chimeric proteins willbe provided in the form of a pharmaceutical composition.

Kits according to this invention can also include instructions, usuallywritten instructions, to assist the user in treating a disease (e.g.,HIV infection or AIDS) with a chimeric bispecific fusion protein. Suchinstructions can optionally be provided on a computer readable medium.

The container(s) in which the protein(s) are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, or bottles. In some applications,chimeric proteins may be provided in pre-measured single use amounts inindividual, typically disposable, tubes or equivalent containers.

The amount of a chimeric bispecific fusion protein supplied in the kitcan be any appropriate amount, depending for instance on the market towhich the product is directed. For instance, if the kit is adapted forresearch or clinical use, the amount of each chimeric protein providedwould likely be an amount sufficient for several treatments.

Certain kits according to this invention will also include one or moreother agents useful in the treatment of disease, e.g. HIV disease. Forexample, such kits may include one or more effective doses of otheranti-virals, immunomodulators, anti-infectives, and/or vaccines.

EXAMPLE 1 Construction of a CD4-SCFv(17b) Encoding Sequence

A gp120-targeted fusion protein, sCD4-SCFv(17b), is constructed bylinking the C-terminus of CD4 (D1D2, 183 amino acid residues) to theN-terminus of the heavy chain of the 17b SCFv, which contains the heavychain at its N-terminus, linked via its C-terminus to the N-terminus ofthe light chain (see schematic diagram of the construct, FIG. 3). The17b SCFv DNA was obtained from R. Wyatt and J. Sodroski, Dana FarberCancer Institute, Boston, Mass. The 17b-MAb producing-hybridoma wasobtained from J. Robinson, Tulane University.

Linkers were chosen to have sufficient length and flexibility to connectthe desired protein segments without inducing unacceptable torsion. Forthe SCFv, the 15 amino acid residue sequence (Gly₄Ser)₃ (designated L2)was chosen, which has been employed successfully for production ofSCFvs. This sequence confers excellent flexibility with minimalaggregation. The linker between the C-terminus of CD4 and the N-terminusof the SCFv (designated L1; SEQ ID NO: 2), is seven repeats of the sameGly₄Ser sequence. Conservative estimates indicate that this 35 aminoacid residue linker is sufficiently long to allow CD4 and SCFv to bindsimultaneously to their respective binding sites on gp120. A schematicof the genetic construct is shown in FIG. 3. A unique BamH I restrictionsite has been introduced within L1 to enable the production ofconstructs with shorter or longer linkers, and especially to providenegative controls (linkers too short, thereby not allowing both the CD4and SCFv moieties of a single molecule to bind simultaneously to theirrespective binding sites on gp120).

The starting CD4 plasmid is pCB-3, which contains the full-length CD4cDNA (including its natural 5′ signal sequence) in the vacciniaexpression plasmid pSC59 (Broder & Berger, J. Virol. 67:913–926, 1993).This plasmid was digested with Stu I, which cuts near the end of the 2nddomain of sCD4, and with Spe I, which cuts within the vector downstreamof the CD4 insert and leaves a 5′ overhang.

Synthetic oligonucleotides (SEQ ID NO: 11) were annealed together torecapitulate the 5′ end of the second half of the Stu I site (CCT) andthe next two bases (CC) of the CD4 cDNA, and to produce an Spe Ioverhang at the 3′ end (this site to be destroyed upon ligation intopCB-3). The oligonucleotide sequence reconstructs the remainder of thesecond domain of CD4 (through ser₁₈₃), and encodes the 37 amino acidintermediate linker (gly₄ser)₆gly₄thr₂ser, followed directly by theuniversal translational termination sequence (UTS) (SEQ ID NO: 6). ABamH I site has been deliberately included within the linker near theend of the third (gly₄ser) repeat, to enable subsequent linkage to the17b SCFv with the exact L1 sequence, and to enable modification oflinker length. The resulting intermediate plasmid is designated pCD1.This construct was confirmed by DNA sequence analysis using standardtechniques. To facilitate subsequent procedures, the sCD4-linkersequence was recloned into a pSC59 derivative lacking a BamH I site,forming intermediate plasmid pCD2.

The starting 17b plasmid containing the 17b SCFv cDNA in a plasmidvector (pmt del 0) was donated by Dr. Richard Wyatt (Dana Farber CancerInstitute, Boston, Mass.). The SCFv cDNA is constructed with the heavychain at the 5′ segment and light chain at the 3′ segment, attached viaDNA encoding the L2 linker (gly₄ser)₃. The 17b SCFv construct has a TPAsignal sequence at the 5′ end, and sequences corresponding to a thrombincleavage site and a hexa-his tag (to facilitate purification) at the 3′end, followed by a stop codon. A comparable construct without thethrombin cleavage site and hexa-his tag can also be produced.

PCR technology was used to attach the 17b SCFv sequence to theCD4-linker sequence in pCD2. Suitable primers are represented in SEQ IDNOs: 7 and 9. The forward (5′) primer (SEQ ID NO: 7) contains a BamH Isite near the 5, end (preceded by an overhang), followed by nucleotidesthat reconstruct the third (gly₄ser) plus four additional (gly₄ser)repeats; this is followed by nucleotides exactly corresponding to thestart of the 17b heavy chain (excluding the 5′ signal sequence,beginning at CAG GTG). The 3′ primer (SEQ ID NO: 9) begins withconvenient restriction sites for cloning into pCD2 (Spe I and others),followed by nucleotides exactly complementary to the 3′ end of the 17bSCFv sequence in pmt del 0 (stop codon, hexa-his tag, and thrombincleavage site).

These primers are used to prime the plasmid vector containing the 17bSCFv sequence in pmt del 0, and the resultant PCR product digested withBamH I plus a restriction enzyme that cleaves at the opposite 3′ end(e.g., Spe I). This digested fragment is then force-cloned into pCD2that has been digested with the same enzymes (BamH I and Spe I). Theresulting plasmid (designated herein as pCD3) contains the finalsCD4-SCFv(17b) construct (with the thrombin cleavage site and hexa-histag) downstream from the strong, synthetic early/late vaccinia promoterin pSC59. There are convenient, unique restriction sites on each side ofthe sCD4-SCFv sequence for possible future cloning steps.

The 17b SCFv cDNA (including the 5′ signal sequence) also has beenexcised from the pmt del 0 vector by restriction enzyme digestion orPCR, and cloned into the vaccinia expression plasmid pSC59 to provide acontrol construct.

EXAMPLE 2 Expression and Purification of CD4-SCFv(17b) Fusion Protein

A. Expression

For small amounts of protein expression, vaccinia expression technologycan be used to produce the sCD4-SCFv(17b) (as well as the control 17bSCFv protein). The plasmid containing the construct in the vacciniaexpression plasmid pSC59 is used to produce a vaccinia recombinant,using standard technology. For such expression, suitable cells (HeLa,BSC-1, etc.) are infected with the recombinant vaccinia virus; afterincubation for 24–36 hours at 37° C., the recombinant protein is presentin the culture supernatant. Initial biochemical and functional studiescan be done with unfractionated supernatant; where necessary, thesCD4-SCFv protein may be purified (see below). Small scale, initialexperiments can be performed with small amounts of material (5–20micrograms, obtained from 1–5×10⁷ cells). The preparation can be scaledup; for such large-scale production, it may be advantageous to employhigher yield technologies for expression of the recombinant proteins(e.g., baculovirus, yeast, or E. coli).

Expression of the pCD1 secreted protein product (the first two domainsof CD4 through ser₁₈₃, plus the 37 amino acid linker) was analyzed.BSC-1 cells were transfected with pCD1 and infected with wild typevaccinia virus, then incubated overnight at 37° C. Supernatants wereanalyzed by Western (immunoblot) analysis, using antibodies against CD4.As expected, the protein encoded by pCD1 migrated slightly more slowlythan standard purified two-domain sCD4 (Upjohn-Pharmacia, Kalamazoo,Mich.).

The pCD3 full-length sCD4-SCFv(17b) (sCD4-17b) fusion protein has beenexpressed and tested similarly, and 17b SCFv domain (as cloned intopSC59) can be examined likewise. The sCD4-17b fusion protein (at least aportion of which is secreted) has the expected molecular size(approximately 55 kD) when analyzed by SDS PAGE and Western blotting.The protein reacted strongly with antibodies against CD4 or the hexa-histag, confirming the presence of these N-terminal and C-terminalmoieties, as well as the correct reading frame.

B. Purification

Expressed fusion protein as constructed above with an amino-terminalhexa-his tag was purified using this molecular tag. The tag enables thespecific binding and purification of the fusion protein by binding tonickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatographymatrix (see, for instance, The QIA expressionist, QIAGEN, 1997). Ahexa-his tag was used in the present examples.

Alternative purification methods include a combination of HPLC andconventional liquid column chromatography (gel filtration; ionexchanger; isoelectric focusing).

C. Primary Characterization

In order to test gp120 binding to the 17b domain of the sCD4-17b fusionprotein, 96-well ELISA plates were first coated with the 13B8.2 anti-CD4MAb (Beckman Coulter, Chaska, Minn., Catalogue no. IM0398), whoseepitope on CD4 overlaps determinants involved in binding to gp120. Theplates were then incubated with either the purified sCD4-17b or controlbuffer. When the chimeric protein was captured this way, the 17b moietyremained available to bind gp120 complexed to sCD4; however the sCD4moiety could not bind free gp120, since it was captured on the plate bythe anti-CD4 MAb that blocks the binding site. The plates were incubatedwith gp120 (IIIB isolate, Ratner et al., Nature 313:277–284, 1985)complexed to sCD4. Binding of gp120 was detected by a polyclonalanti-gp120 antiserum, followed by anti-rabbit IgG conjugated tohorseradish peroxidase. The plates were washed and incubated with ABTSsubstrate, and the oxidized product was quantitated by measuringabsorbance at 405 mm. The results indicated specific binding: absorbancevalues were 0.15 with the sCD4-17b chimeric protein, compared to 0.05with the control buffer.

For testing functionality of the sCD4 region of the chimeric protein,the ELISA plates were first coated with an anti-His tag MAb (QIAGENInc., Valencia, Calif., Catalog no. 34670), then incubated with eitherthe purified chimeric protein or control buffer. With the chimericprotein captured in this way, the sCD4 moiety was available to bind freegp120; however the 17b moiety could not bind gp120 that was notcomplexed to sCD4. The plates were incubated with free gp120, andbinding was detected as detailed above. The results indicated specificbinding: absorbance values were 0.46 with sCD4-17b, compared to 0.05with the control buffer. Thus, the ELISA assays confirmed the expectedfunctional binding properties for each moiety of the chimeric protein:17b moiety bound the gp120/sCD4 complex, and the CD4 moiety bound freegp120.

EXAMPLE 3 HIV-Envelope Neutralization Measurements

A. Vaccinia-Based Reporter Gene Cell Fusion

Env-mediated cell fusion activated by CD4 was measured using thevaccinia-based reporter gene assay (Nussbaum et al., J. Virol.68:5411–5422, 1994). For the experiment shown in Table 2 and FIG. 1,effector cells (HeLa) were transfected with plasmid pG1NT7-β-gal (lacZlinked to T7 promoter), then infected with vaccinia recombinantsencoding either the mutant uncleaved Unc Env or the wildtype (WT) SF162Env (Broder & Berger, Proc. Natl. Acad. Sci., USA 92:9004–9008, 1995).Target cells were created by transfecting NIH 3T3 cells with plasmidpGA9-CKR5, containing the CCR5 cDNA linked to a vaccinia promoter(Alkhatib et al., Science 272:1955–1958, 1996), then infecting thesecells with wild type vaccinia virus WR. Target cells also carry andexpress a bacteriophage T7 RNA polymerase. Prior to fusion assays,transfected cells were incubated overnight at 31° C. to allow expressionof recombinant proteins, then washed.

For each fusion assay, mixtures of effector and target cells (1×10⁵ ofeach cell type per well, duplicate wells) were prepared in the absenceor presence of sCD4 (100 nM final). After 2.5 hours at 37° C., cellswere lysed with NP-40 and β-gal activity was quantitated using standardprocedures (Table 2 and FIG. 1). Relative fusion (specific β-galactivity) was determined from the mean of duplicate samples, andcalculated as WT-Unc.

TABLE 2 Vaccinia-based reporter gene cell fusion assay using soluble CD4Total β-gal (Raw data) Unc Env (Control) WT Env (SF162) Relative fusionduplicates mean duplicates mean (WT - Unc) −sCD4 0.50 0.40 0.45 0.500.40 0.45 0.0 +sCD4 0.40 0.50 0.45 6.60 5.20 5.90 5.45

This vaccinia-based fusion assay can be used to assess the neutralizingability of the herein disclosed bispecific fusion proteins. Theneutralizing ability of MAb 17b was demonstrated to be dependent on theaddition of soluble CD4 as follows (see Table 3 and FIG. 2). Effectorcells were created by co-infecting HeLa cells with a vacciniarecombinant encoding HIV-1 Env (LAV) (Broder & Berger, Proc. Natl. Acad.Sci., USA 92:9004–9008, 1995), and another encoding T7 RNA polymerase.Target cells were created by co-transfecting NIH 3T3 cells with plasmidspYF1-fusin (Feng et al, Science 272:872–877, 1996) encoding CXCR4, andpG1NT7-β-gal (lacZ linked to the T7 promoter). The target cells werethen infected with vaccinia viruses vCB-3 (encoding CD4, standard assay)(Broder et al., Virology 193:483–491, 1993), or WR (wild type virus,sCD4 assay). As background controls, target cells were transfected withpG1NT7-β-gal only (i.e., no coreceptor). Transfected cells wereincubated overnight at 31° C. to allow expression of recombinantproteins, then washed. Effector cells were incubated 30 minutes at 37°C. with the indicated concentration of MAb 17b (Table 3).

For fusion assays, mixtures were prepared between effector and indicatedtarget cells (2×10⁵ of each cell type per well, duplicate wells); in thestandard assay, target cells expressed CXCR4 and CD4, and no soluble CD4added; in the sCD4 assay, target cells expressed CXCR4 alone, andsoluble CD4 was added (200 nM final). After 2.5 hours at 37° C., cellswere lysed and β-gal activity was quantitated. Background control β-galvalues (standard assay, 0.6; sCD4 assay, 0.2), obtained with targetcells lacking coreceptor, were subtracted to give the data presented inTable 3. Data represent percentage of control (no MAb) for each assay.

TABLE 3 MAb-mediated inhibition of fusion assay [17b] Standard AssaysCD4 Assay (μg/ml) β-gal % control β-gal % control none 42.3 100.0 11.89100.0   0.1 39.5 93.4 13.55 113.9   0.5 43.9 103.8 4.66 39.2 1 39.8 94.11.68 14.1 5 50.5 119.4 0 0

The effectiveness of the herein described bispecific fusion proteins forneutralizing fusion is tested in a similar manner, by adding varyingamounts of the bispecific fusion protein, e.g. sCD4-SCFv(17b), to theabove assay. Exogenous sCD4 and SCFv(17b) or other gp120-bindingproteins need not be added, though they can be used as controls asabove, or to determine relative inhibitory efficiencies compared to thebispecific fusion protein. Using this assay, the effects of media fromcontrol cells infected with wild-type vaccinia virus WR, were comparedwith media from cells infected with the recombinant vaccinia virusencoding sCD4-17b. The relative specific β-galactosidase values were23.4 with the control media and <1 with sCD4-SCFv media. Thus, thechimeric sCD4-17b protein strongly inhibited HIV-1 Env-mediated cellfusion.

EXAMPLE 4 Large Scale Production and Analysis of sCD4-17b

To produce large amounts of the sCD4-17b protein, the DNA construct hasbeen transferred to the pET11b plasmid vector (Novagen, Madison, Wis.,Catalog no. 69437-3), which is suitable for high level inducibleexpression in E. coli. This system involves cloning of target genesunder control of strong bacteriophage T7 transcription signal. Onceestablished in a non-expression host bacterial cell, plasmids are thentransferred into expression hosts containing a chromosomal copy of theT7 RNA polymerase gene under lacUV5 control, and expression of therecombinant protein of interest (here, sCD4-17b) is induced by theaddition of IPTG. The expressed protein is produced at a very highlevel, and may constitute more than 50% of the total cell protein in theinduced culture within a few hours after induction. Western blot resultsindicate high level expression of the sCD4-17b from the pET11b plasmid.

The protein produced can be denatured and renatured from inclusionbodies to provide a large quantity of functional sCD4-17b protein. Thisprotein can be used for in vitro studies to test inhibition in assays ofboth Env-mediated cell fusion and HIV infection (p24 production).

In addition, the sCD4-17b protein can be used for in vivo studies. Onein vivo model involves SCID mice reconstituted with human thymus plusliver (Pettoello-Montovani et al., J. Infect. Dis. 177:337–346, 1998);this system will be used to test whether sCD4-17b inhibits (and to whatextent), or prevents, acute HIV-1 infection. This system has beensuccessfully used to demonstrate potent blocking activities of otheranti-HIV agents (e.g., protease inhibitors and reverse transcriptaseinhibitors, and Env-targeted toxins) (Pettoello-Montovani et al., J.Infect. Dis. 177:337–346, 1998).

A second example of an in vivo system for testing sCD4-17b activityinvolves rhesus macaques challenged with SHIV viruses (recombinantviruses containing SIV gag and pol, plus an HIV envelope; L1 et al., J.Virol. 69:7061–7071, 1995). This system will be used to test whether thesCD4-17b protein inhibits (and to what extent), or prevents, acute SHIVinfection.

The effects of sCD4-17b against chronic infection will also be examined,again using the SCID-hu/HIV-1 mouse system and the macaque/SHIV system.

Both in vitro and in vivo study systems also will be used to test thepotency of sCD4-17b protein when used in combination with other anti-HIVagents (e.g., RT and protease inhibitors or other HIV-1 neutralizingMAbs).

The foregoing examples are provided by way of illustration only.Numerous variations on the biological molecules and methods describedabove may be employed to make and use bispecific fusion moleculescapable of binding to two sites on a single protein, and especially twosites on the HIV envelope protein gp120, and for their use in detection,treatment, and prevention of HIV infection. We claim all such subjectmatter that falls within the scope and spirit of the following claims.

1. A neutralizing bispecific fusion protein which binds to two sites ona single gp120 molecule, comprising a first binding domain which bindsto an inducing site on the gp120, thereby exposing an induced epitope ofthe gp120; a second binding domain which binds to and forms aneutralizing complex with the induced epitope of the same gp120; and alinker connecting the first binding domain to the second binding domain,wherein the first binding domain is sCD4, and the second binding domainhas an amino acid sequence comprising at least 90% sequence identity toresidues 244 through 502 of SEQ ID NO:
 3. 2. A protein according toclaim 1, wherein the induced epitope comprises at least one coreceptorbinding determinant of gp120.
 3. A protein according to claim 1, whereinthe inducing site is a gp120 CD4 binding site.
 4. A protein according toclaim 1, wherein the second binding domain binds to at least onecoreceptor binding determinant of gp120.
 5. A protein according to claim1, wherein the linker maintains the second binding domain in bindingproximity to the induced epitope when the first binding domain is boundto the inducing site.
 6. A protein according to claim 5, wherein thelinker is substantially flexible.
 7. A protein according to claim 5,wherein the linker is 15–100 angstroms (Å) long.
 8. A protein accordingto claim 5, wherein the linker is 10–100 amino acid residues in length.9. A protein according to claim 5, wherein the linker comprises at leastone occurrence of an amino acid sequence as represented by SEQ ID NO: 1.10. A protein according to claim 1, wherein the linker comprises atleast one occurrence of an amino acid sequence represented by SEQ IDNO:
 1. 11. A protein according to claim 10, wherein the linker comprisesan amino acid sequence represented by SEQ ID NO:
 2. 12. An isolatednucleic acid molecule encoding the protein according to claim
 11. 13. Anisolated nucleic acid molecule encoding the protein according to claim10.
 14. The protein according to claim 1, wherein the linker is of alength sufficient to maintain the second binding domain in bindingproximity to an SCFv(17b) epitope when sCD4 is bound to gp120.
 15. Theprotein of claim 1, wherein the protein is encoded by a nucleic acidmolecule having a sequence as set forth in SEQ ID NO:
 4. 16. An isolatednucleic acid molecule encoding the protein according to claim
 15. 17. Acomposition comprising the bispecific fusion protein according toclaim
 1. 18. A kit, comprising the composition of claim
 17. 19. The kitof claim 18, further comprising instructions.
 20. The kit of claim 19,wherein the instructions include directions for administering at leastone dose of the neutralizing bispecific fusion protein to a subject inneed of such treatment.
 21. The protein according to claim 1, whereinthe second domain is encoded by an amino acid sequence comprising atleast 95% sequence identity to residues 244 through 502 of SEQ ID NO: 3.22. An isolated nucleic acid molecule encoding the protein according toclaim
 21. 23. The protein according to claim 21, wherein the seconddomain is encoded by an amino acid sequence comprising at least 97%sequence identity to residues 244 through 502 of SEQ ID NO:
 3. 24. Anisolated nucleic acid molecule encoding the protein according to claim23.
 25. The protein according to claim 23, wherein the second domain isencoded by an amino acid sequence comprising at least 98% sequenceidentity to residues 244 through 502 of SEQ ID NO:
 3. 26. An isolatednucleic acid molecule encoding the protein according to claim
 25. 27.The protein according to claim 25, wherein the second domain is encodedby an amino acid sequence comprising at least 99% sequence identity toresidues 244 through 502 of SEQ ID NO:
 3. 28. An isolated nucleic acidmolecule encoding the protein according to claim
 27. 29. The proteinaccording to claim 1, having the amino acid sequence as set forth in SEQID NO:
 3. 30. An isolated nucleic acid molecule encoding the proteinaccording to claim
 1. 31. The nucleic acid molecule according to claim30, wherein the nucleic acid molecule encodes the amino acid sequence asset forth in SEQ ID NO: 3.